The development of modern biotechnology has made artificial insemination a common technique in industrial animals. In particular, artificial insemination with frozen sperm has been established in cattle. Although sexing is an important technique from the industrial viewpoint, a simple and reliable experimental method has been rather difficult to develop in the field of cattle biotechnology.
The following methods have so far been tried in order to sex bovine embryos experimentally.
2.1 Separation and Identification of Y-bearing Spermatozoa
Since females are homozygotic and males are heterozygotic in mammals, including cattle, eggs are sexually homogeneous and spermatozoa consist of two populations. That is, ova fused with Y-bearing spermatozoa become male embryos and ova fused with X-bearing spermatozoa become female embryos. Therefore, if X- and Y-bearing spermatozoa can be separated, female and male embryos can be produced as desired. The following methods have been tried in order to separate X- and Y-bearing spermatozoa.
2.1.1 Separation by Sedimentation Method Utilizing Gravity Force or by Centrifugation in Density Gradients
The X-chromosome comprises approximately 5% of the genome, and the Y-chromosome comprises approximately 3%. This slight difference implies that light spermatozoa are Y-bearing and heavy ones are X-bearing. However, definite separation of X-bearing spermatozoa from Y-bearing spermatozoa is difficult, because the weight difference between X- and Y-bearing spermatozoa, as measured by gravity force is small; the size of spermatozoa naturally fluctuates; and the specific gravity of spermatozoa depends to some extent on the degree of maturity.
2.1.2 Separation by Electrophoresis
Separation by electrophoresis is based on the premise that there is a difference in surface charges between X- and Y-bearing spermatozoa. Generally, charges on the cytoplasmic membrane depend on the amount of sialic acid bound to glycoproteins. During meiosis, however, four spermatids covered by the Sertoli cell are open to each other through cytoplasmic bridges and therefore they form a kind of syncytium. This means that differences in surface charges between X- and Y-bearing spermatozoa are unlikely to occur and therefore, separation by this method is unlikely to be successful.
2.1.3 Discrimination by the Presence of F-body
It is said that when spermatozoa are stained with quinacrine, a fluorescent dye, the Y-chromosome is specifically stained as the F-body in such species as the gorilla and man, and thus X- and Y-bearing spermatozoa can be discriminated when stained with quinacrine. However, this may not hold true for species other than the gorilla or man, because the ratio of spermatozoa having the F-body to those without the F-body is not 50:50. Therefore the Y-chromosome-specific staining technique is theoretically unlikely, i.e., this method seems unlikely to yield accurate results, in principle.
2.1.4 Separation by Flow Cytometry
The X-chromosome is slightly larger than the Y-chromosome. After fluorescent staining of the X- and Y-chromosome, more intense light is emitted from the X-chromosome. This fluorescent staining difference is a basis for separating X-chromosome bearing spermatozoa from Y-chromosome bearing spermatozoa. Insemination of eggs with spermatozoa thus separated enables the production of male or female embryos as desired.
This flow-cytometric method has drawbacks, however, such as the need for the laborious pretreatment of spermatozoas. For example, pretreatment may include dye-staining the spermatozoas and irradiation of the spermatozoa with a laser beam. The spermatozoa may be damaged in such treatments. Micro-insemination, which relies on expensive instrumentation, is necessary for transferring desired spermatozoa identified by flow cytometry. Because of the drawbacks and difficulties of this method, it has not been used commercially yet.
2.2 Anti-HY Antigen Antibody (HY Antibody) Method
A skin graft transplanted from a male to a female in an animal inbred strain is rejected and drops off. The inverse graft from a female to a male is accepted. This phenomenon is considered to be caused by a histocompatibility antigen called the HY antigen which is mapped on the Y-chromosome. The graft rejection reaction is under the control of the cellular immunity. However, anti-HY antibody was also found in the blood of a female rejecting a male graft, indicating the involvement of the humoral immunity.
The HY antigen is expressed at early developmental stages; it has been said to be expressed at the 8 cell stage in mice. There are reports that sexing of murine embryos was possible using the HY antibody derived from mice. It was also claimed that morphological changes of bovine embryos treated with the HY antibody from rats enabled discrimination of male embryos from female ones.
Since the immunogenicity of the HY antigen is considered to be common among species, the HY antibody derived from rats and mice can be applied to cattle. A convenient aspect of using the HY antibody for sexing purposes is that it is not necessary to sample a part of an embryo to determine sexing results.
However, because the HY antigen is not a major histocompatibility antigen but a minor one, its immunogenicity is weak. It is difficult, even impossible, to induce the antibody in some strains. Sometimes the titer of the HY antibody is too low for detection. Determination of the anti-HY antigen/antibody reaction for sexing is not always clear-cut. These drawbacks prevent this method from becoming a reliable sexing method.
2.3 Cytogenetic Method
2.3.1 Method Utilizing the Sex Chromatin
Through inactivation of one X-chromosome of the two X chromosomes in mammalian females, the X-chromosome gene dosage between males and females is equalized. The inactivated X-chromosome is seen in the nucleus as a sex-chromatin. Since the inactivation occurs early in the development, the presence or absence of the sex-chromatin in a part of the trophectoderm microsurgically isolated from a blastocyst would allow prediction of the sex of the embryo. This method is partly successful in mice and rabbits, but cytoplasmic particles obstruct the observation of the sex-chromatin in other domestic animals. This method is not practically used.
2.3.2 Identification of Sex Chromosomes
Direct identification of X,X-chromosomes or X,Y-chromosomes provides a reliable sexing method. The direct identification technique involves bisecting an embryo, with one half being used for embryo transfer and the other, for cytogenetic analysis. Fortunately, all 58 autosomes of cattle are V-shaped telocentric. The X-chromosome of cattle is large and submetacentric, whereas the Y-chromosome of cattle is small and submetacentric. When cattle sample specimens are good, identification of sex chromosomes is easy. When poor metaphase spreads are obtained, the sexing ratios are reduced. Good metaphase spreads are not always obtainable, however, which limits the practical utility of this method.